Antibody-drug conjugates and immunotoxins

ABSTRACT

The present invention relates to conjugates, in particular antibody-drug conjugates and immunotoxins, having the formula I: 
       A-(L-D)p   (I)
 
     or a pharmaceutically acceptable salts or solvates thereof, wherein:
         A is an antibody that selectively binds FAP;   L is a linker;   D is a drug comprising a cytolysin or a Nigrin-b A-chain; and   p is 1 to 10, and to use of such conjugates in the therapeutic treatment of tumors. Methods of producing such conjugates and components for use in such methods are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the continuation of co-pending U.S. patent application Ser. No.15/116,430, filed Dec. 5, 2016, which is the § 371 U.S. National Stageof International Application No. PCT/EP2015/052341, filed Feb. 4, 2015,which was published in English under PCT Article 21(2), which in turnclaims the benefit of GB Application No. 1402006.9, filed Feb. 6, 2014,which is incorporated by reference herein in its entirety.

JOINT RESEARCH AGREEMENT

This application describes and claims certain subject matter that wasdeveloped under a written joint research agreement between TUBEPharmaceuticals, GmbH and ONCOMATRYX BIOPHARMA, S.L. (previouslyONCOMATRIX, S.L.), having an effective date of May 20, 2013.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on October 16,2018, and is -32 kilobytes, which is incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to antibody-drug conjugates (ADCs) andImmunotoxins that target Fibroblast Activating Protein a (FAP), and totheir use in medicine, e.g. in the treatment of certain cancers.

BACKGROUND TO THE INVENTION

Malignant epithelial tumors are the main cancer-related cause of humandeath. These solid tumors frequently exhibit significant stromalreactions such as the so-called “desmoplastic stroma” or “reactivestroma”, which represents 20-60% of total tumor mass and ischaracterized by the existence of large numbers of stromal cells anddense extracellular matrix (ECM). Recent studies have indicated thetumor-promoting roles of stromal cells, as exemplified by vascularcells, immune cells, fibroblasts, myofibroblasts, adipocytes and bonemarrow-derived progenitors (1-6). In particular, considerable numbers ofcancer-associated fibroblasts (CAFs) are frequently observed withintumor-associated stroma of various human cancers, including breast,lung, colon, and pancreas carcinomas (14,15). Interacting coordinatelywith the different components of the stroma, CAFs have the ability topromote neoangiogenesis and tumor growth; CAFs have also been shown ascrucial for the development of aggressive tumors and tumor invasivenessduring cancer progression (16-25); CAFs facilitate the spreading andinfiltration of tumor cells in distant organs, thus contributing toformation of metastases. Importantly, the relevance of stromal cells tothe failure of systemic drug delivery to tumors and to the developmentof drug resistance has also been indicated (7-11).

The identification of cellular and molecular targets abrogatingstromal-tumor cell interactions and thus attenuating tumorigenesis iscurrently one of the most important subjects in translational oncology.Indeed, targeting the peritumoral stroma is a fairly new strategy totreat metastatic tumors, which represent more than 90% of cancer patientmortality: only a few products have obtained therapeutic approval up tonow, most of them being anti-angiogenic drugs (Avastin®; 26).Identifying and targeting other new molecules within the tumormicroenvironment is then essential for increasing the efficacy ofconventional therapies in combination with the stroma-based therapeuticapproaches, and represent a powerful approach for cancer and metastasistreatment (12, 13).

Monoclonal antibody (MAb) - based drugs represent a great promise in thefight against cancer. This is because they allow the treatment to beaimed at a molecular level in a precise and specific way. Theseadvantages, together with their commercial appeal (short developmenttimes, restricted competence and being easily exportable to other cancertypes once they have been approved), have pushed many pharmaceuticalcompanies to invest heavily in the development of new antibody-basedmolecules, as well as in the in-licensing of new molecules ortechnologies from biotech companies.

However, despite the clinical success of therapeutic antibodies, nakedMAbs targeting cell surface tumor antigens rarely present sufficientefficacy on their own. To increase the low activity of the MAbs, novelstrategies are focusing on binding them to toxic molecules. Plant andbacterial toxins as well as small chemotherapeutic molecules can be goodcandidates, since they are very potent and active in very smallquantities.

The field of immunotoxins (ITs) and Antibody-Drug conjugates (ADCs) forthe treatment of cancer has recently experienced a growing developmentactivity by pharmaceutical companies, due to the technological advancesperformed during the last years, aimed at solving the problems theyinitially presented about immunogenicity, undesirable toxicity,production, half-life and resistance.

Immunoconjugates are made of a human, humanized or chimeric recombinantantibody, covalently linked to a cytotoxic drug. The main goal of such astructure is joining the power of small cytotoxic (300 to 1000Da) andthe high specificity of tumor-associated antigen targeted (TAA) MAbs.

The Ab must be very selective to reach the antigen, whose expressionmust be restricted in normal cells. The Ab also must be internalizedefficiently into the cancerous cells.

The cytotoxic agent selected as the effector moiety must kill cells onlyafter internalization and release into the cell cytoplasm. The mostcommonly used payloads in ADCs are DNA-harming drugs such ascalicheamicins, duocarmicins, or microtubule-targeting compounds likeauristatins and maitansinoids.

The Ab-cytotoxic linkers are designed to be stable systemically and torelease the cytotoxic within the target cells.

TAAs are frequently cell membrane proteins that are overexpressed indiseased tissues or at least expressed sufficiently to facilitate theinternalization-activated cytotoxicity. Ideally the antigen presents arestricted expression in normal tissues with a low or absent expressionin vital organs. On top of this, the tumor antigen must be recognizedselectively and with high affinity by an Ab.

In many types of human cancer, fibroblast response is characterized bythe induction of a cell surface protein, Fibroblast Activating Protein a(FAPα), a serine protease of 95 kDa whose expression is highlyrestricted to developing organs, wound-healing and tissue remodeling.

FAP presents the following characteristics:

-   -   Type II membrane glycoprotein with SER-protease activity        (collagenase+DPP)    -   89% human-murine protein homology    -   Tumor stroma-expressed in >90% carcinomas (breast, pancreas,        lung, bladder and colon)    -   Transitory and highly restricted expression in normal adult        tissues during wound-healing and developing organs.    -   FAP(+) fibroblasts located closed to tumor vasculature    -   Very focal expression    -   Internalization    -   Implication in extracellular matrix remodeling, tumor growth and        metastasis.

FAP expression has been recently found in Pancreas tumor cells as wellas tumor-associated stromal fibroblasts. FAP expression was correlatedwith shorter patient survival and worse prognosis, suggesting a possibleFAP-based autocrine/paracrine loop in this type of tumor (32).

During the last 10 years, Kontermann and Pfizenmaier (IZI, University ofStuttgart, Germany) have developed anti-FAP MAb derivatives against bothhuman and murine proteins (27, 28). They have shown in vitro thatanti-FAP scFv immunoliposomes bind specifically FAP+cells and getinternalized (29). In a recent study they demonstrated the anti-tumoraleffect of nanoparticles covered with lipids and anti-FAP scFvs andloaded with TNFα (30).

Treatment with murine MAb FAPS-DM1 immunotoxin induced long lastinginhibition of tumor growth and full regression in pancreas and lungcancer xenograft models, without any intolerance-related effect (31).

Despite these advances, there remains an unmet need for furthertherapeutic strategies for the treatment of tumors, including epithelialtumors, and for components for use in such therapeutic strategies. Thepresent invention addresses these and other needs.

BRIEF DESCRIPTION OF THE INVENTION

Broadly, the present invention relates to anti-FAP antibodies,conjugates thereof and optimised payloads for use in antibody conjugatestrategies. In particular, the present inventors have found thatanti-FAP antibodies as described herein exhibit highly specific binding,and fast and efficient internalisation. Moreover, the present inventorshave found that the A chain of Nigrin b can be isolated and produced inbacterial host cells, yet retains in vitro Ribosome Inactivatingactivity in the absence of the Nigrin-b B-chain and, only onceconjugated to an antibody, exhibits both the ability to translocate intocells and the resulting cytotoxic activity without Nigrin-b B-chain.

The Nigrin-b A-chain described herein and/or cytolysin derivatives areadvantageously conjugated to anti-FAP antibodies for use in thetreatment of tumors.

Accordingly, in a first aspect the present invention provides aconjugate having the formula I:

A-(L-D)_(p)   (I)

or a pharmaceutically acceptable salt or solvate thereof, wherein:

-   -   A is an antibody that selectively binds FAP;    -   L is a linker;    -   D is a drug comprising a cytolysin or a Nigrin-b A-chain; and    -   p is 1 to 10.

In some cases in accordance with this and other aspects of the presentinvention A is a monoclonal antibody or binding fragment thereof thatselectively binds to an extracellular region of human FAP. In some case,A may cross-react to both human and murine FAP. In particular cases Amay comprise heavy chain complementarity determining regions 1-3(CDRH1-3) and light chain complementarity determining regions 1-3(CDRL1-3) having the following amino acid sequences:

-   -   (i) CDRH1: SEQ ID NO: 7 or a variant thereof having up to 1 or 2        amino acid substitutions compared with the sequence of SEQ ID        NO: 7;    -   (ii) CDRH2: SEQ ID NO: 8 or a variant thereof having up to 1 or        2 amino acid substitutions compared with the sequence of SEQ ID        NO: 8;    -   (iii) CDRH3: SEQ ID NO: 9 or a variant thereof having up to 1 or        2 amino acid substitutions compared with the sequence of SEQ ID        NO: 9;    -   (iv) CDRL1: SEQ ID NO: 10 or a variant thereof having up to 1 or        2 amino acid substitutions compared with the sequence of SEQ ID        NO: 10;    -   (v) CDRL2: SEQ ID NO: 11 or a variant thereof having up to 1 or        2 amino acid substitutions compared with the sequence of SEQ ID        NO: 11; and    -   (vi) CDRL3: SEQ ID NO: 12 or a variant thereof having up to 1 or        2 amino acid substitutions compared with the sequence of SEQ ID        NO: 12.

In certain cases, CDRH1-3 comprise the amino acid sequences of SEQ IDNOS: 7-9, respectively and CDRL1-3 comprise the amino acid sequences ofSEQ ID NOS: 10-12, respectively.

In certain cases, A comprises a heavy chain variable region (VH)comprising an amino acid sequence having at least 90%, 95% or 99%sequence identity with the full-length sequence of SEQ ID NO: 5.

In certain cases, A comprises a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO: 5.

In certain cases, A comprises a light chain variable region (VL)comprising an amino acid sequence having at least 90%, 95% or 99%sequence identity with the full-length sequence of SEQ ID NO: 6. Inparticular, A may comprise a light chain variable region (VL) comprisingthe amino acid sequence of SEQ ID NO: 6.

In certain cases, A comprises a heavy chain comprising an amino acidsequence having at least 90%, 95% or 99% sequence identity with thefull-length sequence of SEQ ID NO: 3. In particular, A may comprise aheavy chain comprising the amino acid sequence of SEQ ID NO: 3.

In certain cases, A comprises a light chain comprising an amino acidsequence having at least 90%, 95% or 99% sequence identity with thefull-length sequence of SEQ ID NO: 4. In particular, A may comprise alight chain comprising the amino acid sequence of SEQ ID NO: 4.

In certain cases, A may be a competitively binding anti-FAP antibodythat is structurally different from the anti-FAP antibody moleculesexemplified herein. For example, A may be an anti-FAP antibody moleculethat competes with the anti-FAP IgG1 antibody identified herein as“hu36” for binding to immobilized recombinant human FAP. hu36 has theheavy chain amino acid sequence of SEQ ID NO: 3 and the light chainamino acid sequence of SEQ ID NO: 4. The anti-FAP antibody may, in somecase, bind to the same epitope as hu36. Methods for determining antibodybinding competition and for epitope mapping are well known in the art,see for example “Epitope Mapping by Competition Assay” Ed Harlow andDavid Lane, Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.prot4277.

In accordance with this and other aspects of the present invention, Dmay be a cytolysin. The cytolysin may, in some cases, be a compounddisclosed in WO 2008/138561 A1, the entire contents of which isexpressly incorporated herein by reference (compounds disclosed thereinare also referred to as Tubulysine derivatives). The cytolysin may besynthesised as described in WO 2008/138561. In certain cases, thecytolysin may be as defined in Formula I or Formula IV of WO 2008/138561A1. In certain cases, the cytolysin may be of formula IV:

wherein:

-   -   R² (i) is directly or indirectly attached to linker L or (ii) is        H or is C₁-C₄ alkyl;    -   R⁶ is C₁-C₅ alkyl;    -   R⁷ is C₁-C₅ alkyl, CH₂OR¹⁹ or CH₂OCOR²⁰, wherein R¹⁹ is alkyl,        R²⁰ is C₂-C₆-alkenyl, phenyl, or CH₂-phenyl;    -   R⁹ is C₁-C₅ alkyl;    -   R¹⁰ is H, OH, O-alkyl or O-acetyl;    -   f is 1 or 2;    -   R¹¹ has the following structure:

wherein

-   -   R²¹ is H, OH, halogen, NH₂, alkyloxy, phenyl, alkyl amino or        dialkyl amino;    -   R¹⁶ is H or a C₁-C₆-alkyl group;    -   R¹⁷ (i) is directly or indirectly attached to linker L or (ii)        is CO₂H, CO₂R¹⁸, CONHNH₂, OH, NH₂, SH or a optionally        substituted alkyl, cycloalkyl, heteroalkyl or heterocycloalkyl        group, wherein R¹⁸ is an optionally substituted alkyl,        heteroalkyl or hetercycloalkyl group; and    -   q is 0, 1, 2 or 3;

and wherein the term “optionally substituted” relates to groups, whereinone or several H atoms can be replaced by F, Cl, Br or I or OH, SH, NH₂,or NO₂; the term “optionally substituted” further relates to groups,which can be exclusively or additionally substituted with unsubstitutedC₁-C₅ alkyl, C₂C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ heteroalkyl, C₃-C₁₀cycloalkyl, C₂-C₉ heterocycloalkyl, C₆-C₁₀ aryl, C₁-C₉ heteroaryl,C₂-C₁₂ aralkyl or C₂-C₁₁ heteroaralkyl groups.

In some cases R² is a bond to linker L.

In some cases R¹⁷ is C(O)X, CONHNHX, OX, NHX or SX, wherein X is a bondto linker L.

In some cases linker L may further comprise a spacer.

In some cases the spacer has a chain length of 2 to 30 atoms.

In some cases the spacer comprises or consists of an alkylene (i.e.divalent alkyl) or heteroalkylene (i.e. divalent heteroalkyl) group. Insome cases the spacer comprises or consists of an alkylene oroxyalkylene group.

In some cases the spacer comprises or consists of a group —(CH₂)_(n)— or—(OCH₂CH₂)_(n)—, wherein n 1.

In some cases the spacer comprises or consists of a group—(OCH₂CH₂)_(n)—, wherein n 1. In particular, n may be 1 to 15, 1 to 10,1 to 6, or 2 to 5. For example, n may be 3 or 4.

In some cases the spacer comprises between one and six ethylene glycolunits, e.g. a triethylene glycol.

In some cases the spacer may be directly attached to group R¹⁷, or maybe attached to group R¹⁷ via a bridging group.

In some cases the spacer is attached to group R¹⁷ via a —C(O)X bridginggroup, wherein X is a bond to R¹⁷.

In some cases R¹⁷ is CONHNHX and the spacer is attached to group R¹⁷ viaa —C(O)X bridging group, wherein X represents the bond between thespacer and R¹⁷.

In some cases R¹⁷ is CONHNHX and the spacer is a —(OCH₂CH₂)_(n)—attached to R¹⁷ via a —C(O)X bridging group, wherein n=2, 3 or 4.

In some cases D comprises a cytolysin having the following structure:

In some cases D comprises a cytolysin having the following structure:

In certain cases L comprises an attachment group for attachment to A andprotease cleavable portion. For example, L may comprise avaline-citrulline unit. In particular, L may comprisemaleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate.

In some cases the double bond of the maleimide is reacted with a thiolgroup of a cysteine residue of the antibody A to form a sulphur-carbonbond in order to effect linkage of the linker L to the antibody A.

In some cases -L-D has a structure selected from the group consistingof:

In certain cases -L-D may have the following structure:

In certain cases -L-D may have the following structure:

In accordance with this and other aspects of the present invention pmay, in some cases, lie in the range 1 to 5, e.g. 1 to 4, or 1 to 3. Inparticular cases p may be 1 or 2. In particular, cases p may be 3 or 4.

In accordance with this and other aspects of the present invention D maybe a Nigrin-b A-chain. Preferably, the Nigrin-b A-chain is in theabsence of a Nigrin-b B-chain. The Nigrin-b A-chain may comprise orconsist of the sequence of SEQ ID NO: 13.

In certain cases, the Nigrin-b A-chain may be or may have beenrecombinantly-produced, e.g. in a bacterial host cell. The presentinventors have surprisingly found that Nigrin-b A-chain retains itsactivity (e.g. cytotoxic and/or ribosome inhibiting activity) despiteloss of or alteration of native glycosylation such as is the case whenthe Nigrin-b A-chain is produced recombinantly in a bacterial host cell.

When the conjugate of the present invention comprises a Nigrin-b A-chainas the toxic payload (i.e. D), L may simply be a disulphide bond betweena sulphur atom on A and a sulphur atom on D. Therefore, L may compriseor consist of a disulphide bond.

In a second aspect the present invention provides a conjugate as definedin accordance with the first aspect of the invention for use inmedicine.

In a third aspect the present invention provides a conjugate as definedin accordance with the first aspect of the invention for use in a methodof treatment of a tumor in a mammalian subject.

In some cases the conjugate is for simultaneous, sequential or separateadministration with one or more other antitumor drugs. The one or moreother antitumor drugs comprise a cytotoxic chemotherapeutic agent or ananti-angiogenic agent or an immunotherapeutic agent. In some cases theone or more other antitumor drugs comprise Gemcitabine, Abraxanebevacizumab, itraconazole, carboxyamidotriazole, an anti-PD-1 moleculeor an anti-PD-L1 molecule (for example, nivolumab or pembrolizumab).

In certain cases the conjugate is for use in the treatment of a solidtumor. In particular, the conjugate may be for use in the treatment ofpancreatic cancer, breast cancer, melanoma, lung cancer, head & neckcancer, ovarian cancer, bladder cancer or colon cancer.

In a fourth aspect the present invention provides a method of treating atumor in a mammalian subject, comprising administering a therapeuticallyeffective amount of a conjugate as defined in accordance with the firstaspect of the invention to the subject in need thereof. In some casesthe method may be for treating a solid tumor. In particular, the methodmay be for treating pancreatic cancer, breast cancer, melanoma, lungcancer, head & neck cancer, ovarian cancer, bladder cancer or coloncancer.

In a fifth aspect the present invention provides use of a cytolysin inthe preparation of an antibody-drug conjugate, wherein the antibody isan FAP-specific antibody, e.g., an FAP-specific antibody in accordancewith the eighth aspect of the invention. In some case the use may be ofa cytolysin in the preparation of an antibody-drug conjugate as definedin accordance with the first aspect of the invention.

In a sixth aspect the present invention provides a conjugate of thefirst aspect of the invention for use in the treatment of aninflammatory condition (e.g. rheumatoid arthritis).

In a seventh aspect the present invention provides a method of treatingan inflammatory condition (e.g. rheumatoid arthritis) in a mammaliansubject, comprising administering a therapeutically effective amount ofa conjugate of the first aspect of the invention to the subject in needthereof.

In an eighth aspect the present invention provides an isolated Nigrin-bA-chain in the absence of the Nigrin-b B-chain. The amino acid sequenceof the Nigrin-b A-chain may comprise or consist of the sequence of SEQID NO: 13.

In a ninth aspect the present invention provides use of an isolatedNigrin-b A-chain in accordance with the eighth aspect of the inventionin the preparation of an immunotoxin. In some cases, the immunotoxincomprises a monoclonal antibody conjugated and/or bound to said isolatedNigrin-b A-chain (in the absence of the Nigrin-b B-chain).

In some cases the immunotoxin comprises an antibody, such as amonoclonal antibody, e.g. a human monoclonal antibody, that selectivelybinds FAP. In some cases, the immunotoxin comprises an antibody inaccordance with the tenth aspect of the invention.

In a tenth aspect the present invention provides a monoclonal antibody,e.g. a human monoclonal antibody, that selectively binds FAP and whichcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:3 and a light chain comprising the amino acid sequence of SEQ ID NO: 4.

In an eleventh aspect the present invention provides the antibody of thetenth aspect of the invention for use in medicine. The antibody may befor use in the treatment of an inflammatory condition (e.g. rheumatoidarthritis).

In a twelfth aspect the present invention provides use of a monoclonalantibody in accordance with the tenth aspect of the invention in thepreparation of an antibody-drug conjugate or an immunotoxin.

In a thirteenth aspect the present invention provides a host cellcomprising a vector comprising a polynucleotide that encodes at leastone polypeptide having an amino acid sequence selected from the groupconsisting of: SEQ ID NOS: 1-6 and 13. In some cases the polynucleotidemay comprise the nucleic acid sequence of SEQ ID NO: 14.

In a fourteenth aspect the present invention provides a process for theproduction of a conjugate in accordance with the first aspect of theinvention, comprising:

-   -   (a) derivatising the antibody that selectively binds FAP to        introduce at least one sulphydryl group; and    -   (b) reacting the derivatised antibody with an appropriate        residue (e.g. a cysteine amino acid) on a Nigrin-b A-chain        (absent Nigrin-b B-chain) under conditions which permit the        formation of a disulphide bond linkage between the antibody and        the Nigrin-b A-chain thereby producing the conjugate. The        process may further comprise a step (c) of purifying and/or        isolating the conjugate.

In some cases step (a) may comprise reacting the antibody with4-succynimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene (SMPT),N-succynimidyl 3-(2-pyridyl-dithiopropionate) (SPDP) or methyl4-mercaptobutyrimidate.

In a fifteenth aspect the present invention provides a process for theproduction of a conjugate in accordance with the first aspect of theinvention, comprising:

-   -   (a) linking the antibody that selectively binds FAP to the        linker via a thiol group; and    -   (b) linking the cytolysin to the linker via an appropriate group        on the cytolysin molecule. In some cases, the cytolysin is        linked to the linker via position R₂ or position R₁₇. Steps (a)        and (b) can be performed in either order. In an optional further        step (c), the process may comprise purifying and/or isolating        the conjugate.

The present invention includes the combination of the aspects andpreferred features described except where such a combination is clearlyimpermissible or is stated to be expressly avoided. These and furtheraspects and embodiments of the invention are described in further detailbelow and with reference to the accompanying examples and figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show characterization of humanized scFv hu33 and hu36. FIG.1A) SDS-PAGE analysis of purified scFv fragments. Coomassie staining.R-reducing, NR - non reducing. FIG. 1B) Flow cytometry analysis ofbinding of hu36 (humanized) and mo36 (chimeric) to HT1080-huFAP cells.Bound antibodies were detected with an anti-His-tag antibody (n=2). FIG.1C) ELISA of binding of hu36 scFv and mo36 scFv to immobilizedrecombinant human FAP (coated at 100 ng/ml). Bound antibodies weredetected with an HRP-conjugated anti-Myc-tag antibody.

FIGS. 2A and 2B show ELISA of anti-FAP mo36-IgG1 (circles) and hu36-IgG1(squares) for binding to recombinant human FAP (rhFAP) or controlprotein (BSA) (triangles and inverted triangles, respectively) (FIG.2A). 50 ng protein were coated per well. Bound antibodies were detectedwith HRP-conjugated anti-human IgG-Fc. Flow cytometry analysis ofanti-FAP mo36-IgG1 (triangles and stars) and hu36-IgG1 (squares andcircles) for binding to HT1080-FAP (FIG. 2B). Bound proteins weredetected with a PE-labeled anti-hu IgG-Fc antibody.

FIGS. 3A and 3B show flow cytometry analysis of binding of hu36-IgG1 tostably transfected HT1080 to express human FAP (HT1080-huFAP) (FIG. 3A)and mouse FAP (HT1080-moFAP) (FIG. 3B). Bound antibodies were detectedwith a PE-labeled anti-human Fc antibody.

FIG. 4 shows confocal microscopy of HT1080-FAP cells, incubated withhu36-IgG1 for various times (0, 30 and 60 mins) and stained withFITC-labelled anti-IgG antibody, WGA-TRed (membrane staining), and DAPI(nucleus). The right-hand panels show a merged image of the threestains.

FIG. 5 shows analysis of internalization of hu36-IgG1 by discriminationof cells (n=10-30) showing only membrane staining (PM; open bars), PMand intracellular staining (shaded bars), or only intracellular staining(filled bars). A clear time-dependent internalization is evidenced.

FIG. 6 shows MALDI-Tof profile of recombinant nigrin-b A-chain. Observedmass (Da): 28546.55; Expected mass (Da): 28546.09; Mass deviation: 0.5;Mass Accuracy: 16ppm.

FIG. 7 shows ribosome inactivating protein (RIP) activity of recombinantNigrin-b A-chain (recNgA) tested in rabbit reticulocyte cell-freelysates (RRL) versus native (WT) Nigrin-b. (3a, 3b, 6c, 9c) representdifferent formulations of recNgA.

FIG. 8 shows cytotoxicity of recNgA tested on HT1080-FAP cell linethrough crystal violet viability assay (native Nigrin—diamonds;recombinant Nigrin-b A-chain—squares).

FIG. 9 shows RIP activity of anti-FAP hu36-IgG1-recNgbA immunotoxinconjugates (HSP131-001; crosses) in an RRL assay compared to native (WT)nigrin (triangles) and recombinant Nigrin-b A-chain (recNgA; squares).

FIGS. 10A and 10B show cytotoxic activity of anti-FAP hu36-IgG1-recNgbAimmunotoxin conjugates (HSP131-001; triangles), unconjugated (naked)anti-FAP hu36-IgG1 (squares) and recombinant Nigrin-b A-chain (recNgA;diamonds) on HT1080-WT cell line (FIG. 10A); and HT1080-FAP cell line(FIG. 10B). Fold-change in proliferation is plotted againstantibody/immunotoxin concentration.

FIG. 11 shows the general antibody conjugate structure for acytolysin-conjugated antibody via a vcPABA linker. Attachment of thecytolysin may be via R₁ or R₄ (identified by arrows).

FIG. 12 shows immunodetection of anti-FAP hu36 tumour sections ofpatient-derived xenograft (PDX) mice (pancreatic tumour). Specific Dose-and Time- dependent staining of stroma is observed in subcutaneoustumors from PDX mouse model for pancreas cancer (Panc185)—Single dose (1& 5 mg/kg) of anti-hu/moFAP hu36 IgG1 was administratedintraperitoneally in PDX mice Panc-185; immunodetection was performedwith an anti-human IgG1 secondary antibody—20× scale pictures are shown.Control-48 h: Mice administrated with Vehicle and tumors excised after48 h.

FIG. 13 shows animal weight monitored after treatment withanti-FAP:recNgA immunotoxin at different doses (2.5, 1, 0.5, 0.25, 0.1mg/kg) administrated once a week. Significant weight loss and toxicitywas observed in Group 1 and 2 (2.5 and 1 mg/kg, respectively), similarlyto treatment with 5 mg/kg (not shown); 0.5 mg/kg was the highesttolerated dose when applied as single agent.

FIGS. 14A and 14B show Relative Body weight (FIG. 14A) and Tumor volume(FIG. 14B) measured from patient-derived xenograft mice (PAXF 736)untreated (Vehicle; 10 ml/kg/day; once a week), treated with Gemcitabine(GEM; 150 mg/kg; once a week), or antiFAP:recNgA immunotoxin (OMTX505;0.5/0.25 mg/kg; once a week), or both (OMTX505 (0.25 mg/kg):GEM(150mg/kg)), for 4 weeks (treatment days 1, 8, 15, 22, 29).

FIGS. 15A-15C show ELISA and FACS analysis of ADC471 binding to FAPtarget. ELISA detection of ADC-471 binding to huFAP fusion proteincompared to naked anti-hu/mo FAP hu36 antibody; EC50 values areindicated for HPS124-3 ADC-471 molecule with DAR=3.48 (FIG. 15A); FACSanalysis of binding on HT1080-huFAP, HT1080-wt and HEK293 cells ofHPS131-143-1 (ADC-471; DAR 4), HPS131-124-1 (ADC-467; DAR 1.2) andHPS131-124-3 (ADC-471; DAR 3.48) ADCs (FIGS. 15B and 15C). EC₅₀ valuesare indicated for this latter (FIG. 15B).

FIG. 16 shows Time-lapse immunofluorescence analysis of internalizationcapacity of anti-FAP hu36:cytolysin ADC (ADC-471; HPS131-124-3) onliving HT1080-FAP cells. Left panel: Incubation with naked anti-hu/moFAPhu36 (FITC-AB; green); Right panel: Incubation with ADC-471 (FITC-ADC;green). Time 0, 30, 60, 90min (upper panels): HT1080-FAP cells. Time30min (lower panels): HT1080-wild type cells.

FIGS. 17A and 17B show in vitro cytotoxic effect of anti-hu/moFAP hu36:cytolysin ADCs on HT1080-wt (FIG. 17A) and FAP(+) cells

(FIG. 17B). Cell proliferation arrest was evidenced through crystalviolet staining after 72 h incubation of each compound at aconcentration range from 10⁻⁶ to 10⁻¹²M. Parental TAM334 cytolysin wasused as positive control for unspecific cytotoxicity.

FIGS. 18A and 18B show tumor growth inhibition effect of anti-hu/moFAPhu36:cytolysin ADC candidates. DC471 versus ADC551 (FIG. 18A); ADC471and ADC553 (OMTX705-553) versus ADC558 (OMTX705-558) (FIG. 18B). Vehicleand GEM (Gemcitabine): negative and positive control groups.

DETAILED DESCRIPTION OF THE INVENTION

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below. FAP

As used herein “Fibroblast activation protein”, “fibroblast activatingprotein”, “FAP” and “FAPα” are used interchangeably. The FAP may be anFAP of any mammalian species. In some cases FAP is human FAP (also knownas Seprase, 170 kDa melanoma membrane-bound gelatinase, fibroblastactivation protein alpha or integral membrane serine protease), theamino acid sequence of which is disclosed at UniProt accession No.Q12884 (Version 140, dated 11 Dec. 2013) (SEQ ID NO: 15). In some cases,a molecule that binds FAP (e.g. an antibody molecule or a conjugatethereof) may bind to a region of the extracellular domain of FAP. Theextracellular domain of human FAP comprises residues 26-760 of thefull-length human FAP protein. In some cases FAP is murine FAP (alsoknown as fibroblast activation protein alpha or integral membrane serineprotease), the amino acid sequence of which is disclosed at UniProtaccession No. P97321 (Version 117, dated 11 Dec. 2013) (SEQ ID NO: 16).The extracellular domain of murine FAP comprises residues 26-761 of thefull-length murine FAP protein.

Conjugate

As used herein “conjugate” includes the resultant structure formed bylinking molecules and specifically includes antibody-drug conjugates(ADCs) and immunotoxins (ITs).

Selectively Binds

The terms selectively binds and selective binding refer to binding of anantibody, or binding fragment thereof, to a predetermined molecule (e.g.an antigen) in a specific manner. For example, the antibody, or bindingfragment thereof, may bind to FAP, e.g. an extracellular portionthereof, with an affinity of at least about 1×10⁷M⁻¹, and may bind tothe predetermined molecule with an affinity that is at least two-foldgreater (e.g. five-fold or ten-fold greater) than its affinity forbinding to a molecule other than the predetermined molecule.

Antibody Molecule As used herein with reference to all aspects of theinvention, the term “antibody” or “antibody molecule” includes anyimmunoglobulin whether natural or partly or wholly syntheticallyproduced. The term “antibody” or “antibody molecule” includes monoclonalantibodies (mAb) and polyclonal antibodies (including polyclonalantisera). Antibodies may be intact or fragments derived from fullantibodies (see below). Antibodies may be human antibodies, humanisedantibodies or antibodies of non-human origin. “Monoclonal antibodies”are homogeneous, highly specific antibody populations directed against asingle antigenic site or “determinant” of the target molecule.“Polyclonal antibodies” include heterogeneous antibody populations thatare directed against different antigenic determinants of the targetmolecule. The term “antiserum” or “antisera” refers to blood serumcontaining antibodies obtained from immunized animals.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Thus reference to antibody herein, and

SWG:csw 10/16/18 5351856 CSC/FP7402381 FILED VIA EFS with reference tothe methods, arrays and kits of the invention, covers a full antibodyand also covers any polypeptide or protein comprising an antibodybinding fragment. Examples of binding fragments are (i) the Fab fragmentconsisting of V_(L), V_(H), C_(L) and C_(H)1 domains; (ii) the Fdfragment consisting of the V_(H) and C_(H)1 domains; (iii) the Fvfragment consisting of the V_(L) and V_(H) domains of a single antibody;(iv) the dAb fragment which consists of a V_(H) domain; (v) isolated CDRregions; (vi) F(ab′)₂ fragments, a bivalent fragment comprising twolinked Fab fragments (vii) single chain Fv molecules (scFv), wherein aV_(H) domain and a V_(L) domain are linked by a peptide linker whichallows the two domains to associate to form an antigen binding site;(viii) bispecific single chain Fv dimers (WO 93/11161) and (ix)“diabodies”, multivalent or multispecific fragments constructed by genefusion (WO94/13804; 58). Fv, scFv or diabody molecules may be stabilisedby the incorporation of disulphide bridges linking the VH and VLdomains. Minibodies comprising a scFv joined to a CH3 domain may also bemade.

In relation to a an antibody molecule, the term “selectively binds” maybe used herein to refer to the situation in which one member of aspecific binding pair will not show any significant binding to moleculesother than its specific binding partner(s). The term is also applicablewhere e.g. an antigen-binding site is specific for a particular epitopethat is carried by a number of antigens, in which case the specificbinding member carrying the antigen-binding site will be able to bind tothe various antigens carrying the epitope.

In some cases in accordance with the present invention the antibody maybe a fully human antibody.

Cytotoxic Chemotherapeutic Agents

In some cases in accordance with any aspect of the present invention,the conjugate of the invention may administered with, or foradministration with, (whether simultaneously, sequentially orseparately) one or more other antitumor drugs, including, but notlimited to, a cytotoxic chemotherapeutic agent or an anti-angiogenicagent or an immunotherapeutic agent.

Cytotoxic chemotherapeutic agents are well known in the art and includeanti-cancer agents such as: Alkylating agents including nitrogenmustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide,melphalan (L-sarcolysin) and chlorambucil; 10 ethylenimines andmethylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonatessuch as busulfan;

nitrosoureas such as carmustine (BCNU), lomustine (CCNLJ), semustine(methyl-CCN-U) and streptozoein (streptozotocin); and triazenes such asdecarbazine (DTIC; dimethyltriazenoimidazolecarboxamide);Antimetabolites including folic acid analogues such as methotrexate(amethopterin); pyrimidine analogues such as fluorouracil(5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) andcytarabine (cytosine arabinoside); and purine analogues and relatedinhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine(6-thioguanine; TG) and pentostatin (2′-deoxycofonnycin). NaturalProducts including vinca alkaloids such as vinblastine (VLB) andvincristine; epipodophyllotoxins such as etoposide and teniposide;antibiotics such as dactinomycin (actinomycin D), daunorabicin(daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin(mithramycin) and mitomycin (mitomycin Q; enzymes such asL-asparaginase; and biological response modifiers such as interferonalphenomes. Miscellaneous agents including platinum coordinationcomplexes such as cisplatin (cis-DDP) and carboplatin; anthracenedionesuch as mitoxantrone and antbracycline; substituted urea such ashydroxyurea; methyl hydrazine derivative such as procarbazine(N-methylhydrazine, MIH); and adrenocortical suppressant such asmitotane (o, p′-DDD) and aminoglutethimide; taxol andanalogues/derivatives; and hormone agonists/antagonists such asflutamide and tamoxifen. A further preferred cytotoxic agent isGemcitabine (Gemzar®). A further preferred cytotoxic agent is Paclitaxelbound to human serum albumin (Abraxane®).

Anti-angiogenic agents are well known in the art and include anti-canceragents such as bevacizumab, itraconazole, and carboxyamidotriazole.

Immunotherapeutic agents are known in the art and include, for example,anti-programmed cell death protein 1 (PD-1) antibodies andanti-programmed death-ligand 1 (PD-L1) antibodies, including Nivolumab(MDX1106) and Pembrolizumab (MK-3475).

Pharmaceutical Compositions

The conjugates of the present invention may be comprised inpharmaceutical compositions with a pharmaceutically acceptableexcipient.

A pharmaceutically acceptable excipient may be a compound or acombination of compounds entering into a pharmaceutical compositionwhich does not provoke secondary reactions and which allows, forexample, facilitation of the administration of the conjugate, anincrease in its lifespan and/or in its efficacy in the body or anincrease in its solubility in solution. These pharmaceuticallyacceptable vehicles are well known and will be adapted by the personskilled in the art as a function of the mode of administration of theconjugate.

In some embodiments, conjugates of the present invention may be providedin a lyophilised form for reconstitution prior to administration. Forexample, lyophilised conjugates may be re-constituted in sterile waterand mixed with saline prior to administration to an individual.

Conjugates of the present invention will usually be administered in theform of a pharmaceutical composition, which may comprise at least onecomponent in addition to the conjugate. Thus pharmaceutical compositionsmay comprise, in addition to the conjugate, a pharmaceuticallyacceptable excipient, carrier, buffer, stabilizer or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the conjugate.The precise nature of the carrier or other material will depend on theroute of administration, which may be by bolus, infusion, injection orany other suitable route, as discussed below.

For intra-venous administration, e.g. by injection, the pharmaceuticalcomposition comprising the conjugate may be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles, such as Sodium Chloride Injection, Ringer'sInjection, Lactated Ringer's Injection. Preservatives, stabilizers,buffers, antioxidants and/or other additives may be employed as requiredincluding buffers such as phosphate, citrate and other organic acids;antioxidants, such as ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3′-pentanol; and m-cresol); low molecularweight polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone;amino acids, such as glycine, glutamine, asparagines, histidine,arginine, or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose or dextrins; chelating agents,such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol;salt-forming counter-ions, such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG).

Subject

The subject may be a human, a companion animal (e.g. a dog or cat), alaboratory animal (e.g. a mouse, rat, rabbit, pig or non-human primate),a domestic or farm animal (e.g. a pig, cow, horse or sheep). Preferably,the subject is a human. In some cases the subject may be a humandiagnosed with or classified as being at risk of developing a cancer,e.g., an epithelial tumor. In certain cases the subject may be alaboratory animal, e.g., a mouse model of a cancer. In certain cases thesubject may be a mammal (e.g. a human) that has been diagnosed with orclassified as being at risk of developing an inflammatory condition,such as rheumatoid arthritis (RA). In particular, the subject may be ahuman having RA.

Cancer

The anti-FAP conjugates described herein find use in the treatment of atumor in a mammalian subject. The tumor may be a solid tumor. Inparticular, the tumor may be a pancreatic cancer, breast cancer,melanoma, lung cancer, head & neck cancer, ovarian cancer, bladdercancer or colon cancer.

Inflammatory Condition

In some cases in accordance with the present invention, the anti-FAPantibody or the antibody drug conjugate may be for use in the treatmentof an inflammatory condition. FAP expression has been reported infibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA)patients (see, e.g., Bauer et al., Arthritis Res. Therp. (2006):8(6);R171). The present inventors believe that the anti-FAP antibodiesdescribed herein, and/or conjugates thereof described herein, are ableto ameliorate RA and/or symptoms of RA.

The following is presented by way of example and is not to be construedas a limitation to the scope of the claims.

EXAMPLES Example 1 Production of Anti-FAP Antibodies

Anti-FAP scFvs selected by phage display from an immunized FAP^(−/−)knock-out mouse have been described previously (28). Two scFvs, “MO36”and “MO33”, cross-reactive for human and murine FAP (28) were convertedinto full-length IgG for subsequent characterisation studies and forgeneration of immunotoxins and ADCs. These scFv (scFv33 and scFv36) wereused to generate chimeric antibodies, fusing heavy and light chainconstant domains to VH and VL, respectively. In addition, both werehumanized by CDR grafting and tested for binding to FAP-expressing cellsand recombinant FAP in comparison to the parental scFv. From thiscomparison, the best binder was used to generate full-length IgG. AllscFvs were produced in E. coli and purified by IMAC, IgGs were producedin mammalian cells (CHO) using the Lonza GS expression vectors pEE6.4and pEE14.4 developed for antibody production. Features of the scFvs aresummarized in Table 1.

TABLE 1 antibodies, specificities, subclass, and vectors used asstarting material Vl Plasmid Format Species Antigen Clone SubclassVector DNA # scFv mouse hu/mo FAP mo33 lambda pAB1 376 scFv mouse hu/moFAP mo36 kappa pAB1 277 scFv humanized hu/mo FAP hu33 lambda pAB1 1214scFv humanized hu/mo FAP hu36 kappa pAB1 1215

All scFvs were bacterially produced in E.coli TG1 and purified from theperiplasmic extracts of 1 L cultures by IMAC. Both humanized antibodies(scFv hu33 and hu36) were purified in soluble form with yields ofapproximately 0.6 mg/L culture. In SDS-PAGE the proteins migrated withthe expected size of approximately 30 kDa (FIG. 1A). Purity wasestimated to be >90%. In flow cytometry experiments using HT1080 cellsexpressing human FAP (stable transfectants), a similar binding wasobserved for scFv hu36 and mo36 scFv, which was also produced inbacteria (not shown). EC₅₀ values were in the low nanomolar range. Somedifferences were observed at higher concentrations (FIG. 1B). scFv hu33showed no binding or only marginal binding in these experiments. Furtherdevelopment therefore focused on hu36. Binding of hu36 scFv was alsoobserved by ELISA with recombinant human FAP (extracellular region aa26-760; R&D systems), although binding was somewhat weaker than thatseen for mo36 scFv (FIG. 1C).

Plasmids corresponding to full length IgG1 antibodies were generated andtransfected into CHO cells for production of antibodies in Lonza's CHOexpressing system with yields of approximately 1 mg/L of cell culture(lab scale). Antibodies were purified from cell culture supernatant byprotein A chromatography. Purified proteins were characterized bySDS-PAGE and size exclusion chromatography. Bioactivity was analyzed byELISA using recombinant FAP and detection of bound antibodies withHRP-conjugated anti-human IgG antibodies. Cell binding was analyzed byflow cytometry using HT1080-FAP cell line.

Results:

Plasmids generated (and sequenced):

mo36 IgG1: pEE14.4 mo36-IgG1 OCMTX001p (chimeric anti-FAP IgG1) hu36IgG1: pEE14.4 hu36-IgG1 OCMTX002p (humanized anti-FAP IgG1)

Example 2 Characterisation of Anti-FAP Antibodies

The amino acid sequences of humanized anti-FAP IgG1 hu36 (hu36-IgG1)heavy chain (HC) and light chain (LC), respectively are shown below:

Anti-FAP hu36-IgG1-HC:

(SEQ ID NO: 1)

GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK aa 449 MW of processed HC 49,069Theoretical pI 8.69Potential glycosylation site (double underlined): N297Mutations leading to ADCC and CDC deficiency are shown in bold italics(see also WO 99/58572) Signal sequence is shown boxedVH domain is underlined; CDRH1-H3 are shown in bold and curvedunderlined.Anti-FAP hu36-IgG1-LC:

(SEQ ID NO: 2)

NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC aa 218 MW of processed HC 23,919 theoretical pI 7.77signal sequence is boxedVL domain is underlined; CDRL1-L3 are shown in bold and curvedunderlined. hu36-IgG1-HC—without signal sequence: (SEQ ID NO: 3)

GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hu36-IgG1-LC—without signal sequence:(SEQ ID NO: 4)

NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC hu36-VH: (SEQ ID NO: 5)QVQLVQSGAEVKKPGASVKVSCKASGYTFTENIIHWVRQAPGQGLEWMGWFHPGSGSIKYNEKFKDRVTMTADTSTSTVYMELSSLRSEDTAVYYCARHGGTGRGAMDYWGQGTLVTVSS hu36-VL:(SEQ ID NO: 6)DIQMTQSPSSLSASVGDRVTITCRASKSVSTSAYSYMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSRELPYTFGQGTKLEIKR hu36-CDRH1: (SEQ ID NO: 7)ENIIH hu36-CDRH2: (SEQ ID NO: 8) WFHPGSGSIKYNEKFKD hu36-CDRH3:(SEQ ID NO: 9) HGGTGRGAMDY hu36-CDRL1: (SEQ ID NO: 10) RASKSVSTSAYSYMHhu36-CDRL2: (SEQ ID NO: 11) LASNLES hu36-CDRL3: (SEQ ID NO: 12)QHSRELPYT Parameters of the full hu36-IgG are as follows:Total length of full-length IgG (aa): 1,334Calculated molecular mass of full-length IgG: 145,922Calculated extinction coefficient of full-length IgG: 209,420Abs 0.1% (=g/l) 1.435 theoretical pI: 8.60 potential glycosylation site:N297

Purified chimeric and human anti-FAP antibodies mo36 and hu36 wereanalyzed in ELISA for binding to recombinant FAP. Both anti-FAPantibodies showed specific and strong binding to recombinant FAP withsimilar EC₅₀ values (around 5 nM) (FIG. 2A). Furthermore, bothantibodies showed binding to HTP1080-FAP expressing human FAP on theircell surface (FIG. 2B). The humanized IgG gave stronger signals comparedwith the chimeric IgG, however, with similar EC₅₀ values. The humanizedhu36 anti-FAP antibody was able to cross-react to both human and murineFAP as shown by FACS analysis (FIGS. 3A and 3B). Hu36-IgG1 bound in aconcentration-dependent manner to both cell lines with subnanomolar EC50values (0.33 and 0.25 nM).

For scale-up the antibody constructs were cloned in GS double vectors(pEE14.4). The DNA plasmids were transformed, amplified, and transientlytransfected into CHOK1SV cells for expression evaluation at a volume of200 ml. In a second step the antibodies were transiently expressed in5-10 L large scale cultures. Clarified culture supernatant was purifiedusing one-step Protein A chromatography. Product quality analysisthrough SE-HPLC, SDS-PAGE and LAL was carried out using purifiedmaterial at a concentration of 1 mg/ml, alongside an in-house humanantibody as a control sample.

The purified protein samples were filtered through a 0.2 μm filter andanalysed by SE-HPLC chromatograms. The antibodies were purifiedto >98.8%. The endotoxin levels were <0.5 EU/mg.

All purified proteins were analyzed by SDS-PAGE in reducing andnon-reducing conditions (data not shown).

Purified proteins hu36-IgG and mu36-IgG were characterized by SDS-PAGEand size exclusion chromatography. Bioactivity was analyzed by ELISA,using recombinant FAP and detection of bound antibodies withHRP-conjugated anti-human IgG antibodies. Cell binding was analyzed byflow cytometry, using HT1080-FAP cell line. Melting points weredetermined by dynamic light scattering using a zetasizer nano.Affinities were determined by QCM using an Attana A100. Internalizationstudy was performed by indirect immunofluorescence confocal microscopyon permeabilized cells, detecting bound and internalized antibodies witha FITC-labeled secondary antibody.

The full-length IgG1 purified antibodies were successfully produced atboth lab scale and large scale, for the generation of immunoconjugates.A summary of antibody properties is shown in Table 2. The antibodiesretained their specificity, as shown by ELISA and flow cytometryexperiments. The antibodies bound FAP-expressing cells with subnanomolarEC₅₀ values. Affinities, as determined by QCM, were comparable with thatof parental antibodies. QCM measurements indicated the contribution ofavidity effects to high-affinity binding. Thermal stability differedbetween the different IgGs (77-80° C.)

Rapid internalisation was shown for hu36-IgG1 (humanized anti-FAPantibody) on HT1080-FAP cells (see FIGS. 4 and 5).

TABLE 2 Summary of antibody properties antibody mo36-IgG1 hu36-IgG1antigen hu and mo FAP hu and mo FAP isotype γ1*/κ γ1*/κ IgG typechimeric humanized plasmid OCMTX001p OCMTX002p purity (SEC) minor ✓aggregates Tm (DLS) 77° C. 80° C. EC₅₀ ELISA 3 nM (rhFAP) 3 nM (rhFAP)EC₅₀ FACS 0.5 nM (HT1080- 0.3 nM (HT1080- huFAP) huFAP) 0.2 nM (HT1080-moFAP) binding to n.d. + primary tumor fibroblasts binding rhFAP: rhFAP:constants K_(D) K_(D)1 = 112 nM K_(D)1 = 218 nM (QCM) K_(D)2 = 0.6 nMK_(D)2 = 0.4 nM internalization n.d. HT1080-FAP 30-60 min γ1* =deficient for ADCC and CDC (see Amour et al., 1999; Richter et al.,2013).

Anti-FAP IgG1 In Vivo Binding.

Anti-FAP IgG1 hu36 was administrated intraperitoneally topatient-derived xenograft mice for pancreas cancer at a single dose of 1and 5 mg/kg. Tumors were excised after 12, 24, and 48 h administration,formalin-fixed and paraffin-embedded. Immunodetection of anti-FAP hu36was performed with an anti-human IgG secondary antibody. FIG. 12 showsthe specific dose- and time-dependent staining of stroma, only in tumorsamples from treated mice.

Example 3 Nigrin-b A-Chain

In order to avoid side effects of free toxin that could be released inthe bloodstream and to reduce potential immunogenicity of the RIP toxin,as extensively described with ricin, the enzymatic domain of Nigrin b,the A chain, was cloned and expressed in bacteria. The present inventorshypothesized that, if the A chain produced in bacteria was able toretain its activity, it would not be able to enter the cells, unlessconjugated to a vehicle molecule, such as an antibody.

Production

Nigrin-b A-chain was synthetized taking into account codon optimizationfor bacterial expression and the synthetized gene was cloned in twodifferent vectors, Nigrin_pET30b-3 and Nigrin_pET33b-1 (+/−His tag) forexpression in two different E. coli strains, E. coli BLR(DE3) and E.coli HMS174(DE3). Different culture media were used to check differentexpression conditions. Process purification was established using CaptoQ chromatography and SP Sepharose High Performance. Purified recombinantNigrin-b A-chain (recNgA) was formulated at 5 mg/ml in PBS 1× pH7.4, DTT0.5 mM, glycerol 10%. Endotoxin levels were <1EU/mg of Nigrin and thepurity >99% in monomeric form.

Eldman N-terminal sequencing revealed that N-terminal end of recNgAcorresponded to the expected sequence.

Recombinant Nigrin-b A-chain amino acid sequence: (SEQ ID NO: 13)MIDYPSVSFNLDGAKSATYRDFLSNLRKTVATGTYEVNGLPVLRRESEVQVKSRFVLVPLTNYNGNTVTLAVDVTNLYVVAFSGNANSYFFKDATEVQKSNLFVGTKQNTLSFTGNYDNLETAANTRRESIELGPSPLDGAITSLYHGDSVARSLLVVIQMVSEAARFRYIEQEVRRSLQQATSFTPNALMLSMENNWSSMSLEIQQAGNNVSPFFGTVQLLNYDHTHRLVDNFEELYKITGIAILLFRC SSPSND

The recombinant Nigrin-b A-chain has the following characteristics:

-   -   Number of amino acids: 256    -   Molecular weight: 28546.0    -   Theoretical pI: 5.45

The nucleotide sequence encoding recombinant Nigrin-b A-chain is asfollows:

(SEQ ID NO: 14) atagactatc cctccgtctc cttcaacttggatggagcca agtcggctac atacagggac ttcctcagca acctgcgaaa aacagtggcaactggcacct atgaagtaaa cggtttacca gtactgaggc gcgaaagtga agtacaggtcaagagtcggt tcgttctcgt ccctctcacc aattacaatg gaaacaccgt cacgttggcagtagatgtga ccaaccttta cgtggtggct tttagtggaa atgcaaactc ctactttttcaaggacgcta cggaagttca aaagagtaat ttattcgttg gcaccaagca aaatacgttatccttcacgg gtaattatga caaccttgag actgcggcga atactaggag ggagtctatcgaactgggac ccagtccgct agatggagcc attacaagtt tgtatcatgg tgatagcgtagcccgatctc tccttgtggt aattcagatg gtctcggaag cggcaaggtt cagatacattgagcaagaag tgcgccgaag cctacagcag gctacaagct tcacaccaaa tgctttgatgctgagcatgg agaacaactg gtcgtctatg tccttggaga tccagcaggc gggaaataatgtatcaccct tctttgggac cgttcagctt ctaaattacg atcacactca ccgcctagttgacaactttg aggaactcta taagattacg gggatagcaa ttcttctctt ccgttgctcctcaccaagca atgat 

Materials

-   -   Nigrin_pET30b-3 genetic construct.    -   Escherichia coli (Migula) Castellani and Palmers BLR(DE3)    -   Culture media: auto induced medium (AIM)    -   Extraction culture buffer: Glycine/NaOH 10 mM, Leupeptine 1        μg/ml, Pepstatine 1 μg/ml, pH 9.5.    -   Extraction supernatant buffer Tris-HCl 50 mM, NaCl 200 mM, MgCl₂        2 mM, leupeptine 1 μgml⁻¹, pepstatine 1 μgml⁻¹, lysozyme 0.1        mgml⁻¹, pH8.0.    -   Dialysis solution: Citric acid/NaOH 25 mM pH5.0. -Capto Q FPLC:        Equilibration buffer A: Glycine/NaOH 50 mM pH9.5. Elution buffer        B: Glycine/NaOH 50mM pH9.5, NaCl 1 M.    -   Pooled fractions from Capto Q step (+80 ml extraction).    -   SP Sepharose HP FPLC: Equilibration buffer A: Citric acid 25 mM        pH4.0.Elution buffer B: Citric acid 25 mM pH4.0, NaCl 1 M.

Methods

E. coli BLR(DE3) holding expression Nigrin_pET30b-3 cultivated in 1Lformat of Auto Inducible Medium (AIM) with 30 μgml⁻¹ Kanamycin. Proteinexpression was triggered by lactose activation and glucose depletionafter about 3-4 hours of growth. Then, the temperature was lowered to20° C. for an overnight duration.

For extraction, each cell pellet was initially resuspended in 80 ml ofextraction buffer per liter of culture, and 3 cycles of 7 minutesdisintegration at 1100-110 Bar were performed after 30 minutes ofincubation at 8° C. under shaking. Then the extract underwent 60 minutescentrifugation at 15,900g, 8° C. The supernatant was the purification'sstarting material.

Capto Q FPLC: 160 ml of extracted product from 81 culture were loadedinto 160 ml Capto Q and equilibrated using 4CV of equilibration bufferand washed with 15 CV of equilibration buffer. Elution was carried inthree steps: 15 CV at 1.5 mS/cm (7.6% B); 20 CV at 23.8 mS/cm (18.9% B);20 CV 100% B.

Dialysis was performed at the following conditions: 650 ml of theproduct were dialyzed in 4×5Lbathsin in citric acid/NaOH 25 mM pH5.0,cut-off 6-8000 Da. Dialysis factor 3500, <24 h. After dialysis, a 30minutes centrifugation at 20,500 g and 8° C. allowed to separate solublefrom insoluble fractions. SDS-PAGE was performed on the total andsoluble fractions both pre and post dialysis (10 μl loaded on SDS-PAGE).The eluent was dialysed into PBS pH7.4 and filtered T=0.22 μm using 2×20cm² EKV filters.

SP Sepharose HP: 610m1 of dialyzed pool of Capto Q in Citric acid 25 mMpH5.0 were loaded into 240m1 SP Sepharose High Performance with 4 CV ofequilibration buffer and washed with 15CV of equilibration buffer andeluted at 25 Cv gradient to 20% B; 4 CV step of 100% B.

Pooled fractions from SP Sepharose HP step were dialysed in PBS pH7.4,DTT 0.5 mM (5×4L baths, pooled fractions of 950 mL at 0.97 mg/ml). Cutoff was 6-8000 Da, dialysis factor was −3130, time >24 h. Afterwards a30 min centrifugation at 20,55 g and 8° C. allowed to separate solublefrom insoluble fractions. 10% glycerol was added afterwards.

Finally the eluent was dialysed into PBS pH7.4 (5 baths ˜3100) andfiltered φ=0.2 μm, then the recNg b A batch was snap frozen at −80° C. ASEC in Semi-Preparative 5200 Superdex was later carried out.

Size exclusion chromatography and mass spectrometry analysisdemonstrated monomeric and purification status of the obtainedrecombinant nigrin-b A-chain (recNgA) (FIG. 6).

Stability studies were performed to evaluate pH and temperature effecton nigrin-b A-chain protein itself and its activity. recNgA is stable atpH ranging from 5 to 9, and in presence or not of glycerol (from 10 to45%) (data not shown).

Activity

The ribosome-inactivating protein (RIP) activity of recombinant Nigrin-bA-chain was tested in rabbit reticulocyte cell-free lysates: IC₅₀ valueobtained was similar to native nigrin-b and within 2.5 to 25 pM range(see FIG. 7). Thus, the A chain from Nigrin-b, expressed as arecombinant protein in bacteria, maintains its enzymatic activity,supporting that glycosylation is not required for RIP activity ofNigrin-b A-chain.

RecNgA retains its activity in rabbit reticulocyte cell-free lysates ifstored frozen at (−80° C.) and below 3 freeze-thaw cycles (not shown).

The cytotoxic activity of recNgA was tested on cell cultures throughcrystal violet-based viability assay. recNgA, lacking the B chain totranslocate within cells, presents a 100 to 1000 less toxic activitythan native Nigrin-b, as shown in FIG. 8. Native nigrin b showed anIC₅₀≈2×10⁻⁸M (similar to previous published data; see 33), while recNgAshowed an IC₅₀≈2×10⁻⁶M.

Previously published studies showed that native Nigrin b presents higherRIP activity than Ricin in RRL assay, while it is much less toxic(30-10,000 time, approximately) in cells or in vivo (see IC₅₀ and LD₅₀values in Table 3).

Upon removing of B chain, Ricin A chain loses activity in both RRL assayand cytotoxicity assay. Unexpectedly, Nigrin b A chain, generated forthe first time in this present invention, only loses activity in cellcytotoxicity assay, while it was even increased in

RRL assay with respect to native Nigrin b. These data were suggestingthat, in the case of Ricin, removing B chain was affecting not onlybinding and translocation of A chain, but also its RIP activity, whilethis was not the case for Nigrin b A chain that retains and evenincreases its RRL activity with respect to its native counterpart. As aresult, Nigrin b A chain is 50 times more active than the Ricin A chainin RRL.

Consequently, upon conjugation, Nigrin b A chain conjugates presenthigher cytotoxic activity (IC₅₀ within pM range) than Ricin A chainconjugates (nM range) (data not shown).

TABLE 3 In vitro and in vivo activity data for Ricin and Nigrin (nativeand A chain). Rabbit Lysate HeLa Cells IC₅₀ Mouse LD₅₀ IC₅₀ (pM) (pM)(μgkg⁻¹) Nigrin b 30 27,600.00 (20-2300 nM; 12,000.00 dpt cell line)Nigrin b 6.5 750,000.00 ND A chain (HT1080-FAP) 300,000.00 (HT1080)Ricin 100 0.67 3.00 Ricin A 300 260,000.00 (T ND chain cells)(Inventors' Own data - Nigrin b A chain; see also Ferreras J. M. et al.,Toxins, 3: 420, 2011; Svinth M. et al., BBRC, 249: 637, 1998)

Example 4 Conjugation of Nigrin-b A-Chain to Anti-FAP Antibodies

For immunoconjugates containing RIPs to exhibit maximal cytotoxicity theRIP must be released from the targeting vehicle in fully active form,which requires avoiding steric hindrance (34)). The disulfide bond isthe only type of linkage that fit this criterium (35, 36). This bondallows conjugation using reagents for the introduction of freesulfhydryl groups such as N-succynimidyl 3(2-pyridyl-dithiopropionate)(SPDP) and 4-succynimidyloxycarbonyl-α-methyl-α(2-pyridyl-dithio)toluene (SMPT). Immunotoxins consisting of mAbscovalently bound to toxins by hindered disulfide linkers, often labeledas second generation immunotoxins, are stable, long lived and displaypotent cytotoxicity to target cells (37).

SPDP has already been used in the making of immunotoxins (ITs)containing nigrin b (38, 39). Moreover SMPT protects the disulfide bondfrom attack by thiolate anions, improving in vivo stability of thelinkage (40, 41).

Material

-   -   Recombinant nigrin b A chain in PBS, pH7.4, 10% glycerol, 0,5 mM        DTT, 4.92gl⁻¹, stored at 5° C.    -   5,5′-dithio-bis-(2-nitrobenzoic acid)    -   GE PD MiniTrap G-10 desalting columns.    -   0.2 μm 28 mm sterile Minisart filters.    -   Sciclone ALH 3000 workstation.    -   Sarstedt Microtest Plate 96-Well Flat Bottom, ref n° 82.1581.

Methods

Dithiothreitol (DTT, Cleland's reagent) is a redox agent that will beused to free the thiol groups present in the protein sample. Once saidgroups have been freed and so are available for reacting5,5′-dithio-bis-(2-nitrobenzoic acid) (Ellman reagent) will be added.Ellman reagent disulphide bridge will be cleaved and the 2 resultingthio-nitrobenzoate molecules (TNB) will attach to the protein at thethiol group sites. To titrate the TNBs absorbance values will be takenat λ=412 nm, a wavelength at which DTT is not absorbed, rendering theconcentration of thiol groups. The proportion of these with theconcentration of the protein taken from its absorbance at λ=280 willyield the number of free thiol groups per protein molecule.

Direct thiol titration was performed as follows: 204 μl recNg b A weredissolved in 796 μl 20 mM phosphate 250 mM NaCl 1 mM EDTA pH 7.0 (assaybuffer) (1.0033 gl⁻¹=final concentration). Ellman reagent was dissolvedin phosphate 0.2 M at 3gl⁻¹. For both buffers monobasic and dibasicsodium phosphate were added in a 1.61 to 1 mass proportion. PH wasadjusted at room temperature and buffers were filtered. 100 ml Ellmanbuffer and 500 ml assay buffer were prepared. Ellman reagent wascompletely resuspended rather than weighed.

The recNgA sample was incubated in the presence of 4.8 mM DTT at roomtemperature for 30 min. The recNgbA sample was then purified in thecolumn and the first 10 ml of the eluent aliquoted (V=0.5 ml). The A₂₈₀of the aliquots was taken and the two most concentrated mixed. A₂₈₀ wastaken again. 10 μl of 3 gl⁻¹ DTNB were added and A₄₁₂ measured after 2min (n=1), using Ellman diluted in assay buffer in the sameconcentration as a blank (n_(b)=3) . Readings belonged to the 0.1-3 AUlinear range. Protein solutions were pipetted right beneath the meniscusafter vortexing. 100 μl were pipetted per well. The results of thisstudy show that the thiol group belonging to recNgA's single cysteineresidue is free and available for reaction, not being blocked by itstertiary structure. This will allow recNgbA to be conjugated using alinker that requires a hindered inter-chain disulfide bond.

It is well established that immunoconjugates which containribosome-inactivating proteins exhibit maximal cytotoxicity only whenthe toxin molecule is released from the targeting vehicle in a fullyactive form. The separation of the RIP molecule from the carrier isrequired to avoid steric hindrance and to allow an effectivetranslocation of the toxin into the cytoplasm (34)). At present, thedisulfide bond is the only type of linkage which appears to fit thesecriteria (36).

The coupling of two different protein macromolecules, that results inheterodimer formation, requires that each protein is modified prior tomixing them to react. In the case of the A chains of type 2 RIPs, themodification is limited to the reductive cleavage of the native cysteineresidue that links the active (A) and the binding (B) chains of themolecule.

For IgG molecules, this is not possible because cysteine residues areinvolved in maintaining the tertiary and/or quaternary structure of theprotein, so that it is not possible to reduce them without loss of thespecific protein functions. Moreover, presumably some of the cysteineresidues are not sterically accessible, as it was demonstrated by the 10thiols groups per immunoglobulin that had to be generated for an optimalconjugation to an activated RIP (42).

For these reasons, in most IgG molecules, thiol groups are chemicallyinserted using hetero-bifunctional reagents, and several methods havebeen developed in order to generate hetero-conjugates avoiding orreducing to a minimum the formation of homopolymers. In most cases, thereagents used to introduce thiol groups react with amino groups, formingamide or amidine bonds. Amino groups are reactive, abundant and, in alimited way for most proteins, expendable. That is, a limited number ofamino groups can be modified without diminishing the biological activityof the protein (36).

The most commonly used reagents for the introduction of free sulphydrylgroups are N-succynimidyl 3-(2-pyridyl-dithiopropionate) (SPDP) and4-succynimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene (SMPT),that introduce 2-pyridyl disulphide groups into the protein by reactingwith amino groups to form neutral amides, and methyl4-mercaptobutyrimidate (2-iminothiolane.Traut's reagent) that introducesmercaptobutyrimidoyl groups, reacting to form charged amidines, thuspreserving the positive charge of the derivatized amino acid (36, 41).

SPDP and SMPT introduce hindered disulphide bond, while 2-iminothiolane-SH must be protected by reacting it with 5,5′-dithiobis-2-nitrobenzoicacid (Ellman's reagent). The reaction with Ellman's reagent is also usedfor the quick measurement of protein sulphydryl groups (43, 44).

SMPT has a methyl group and a benzene ring attached to the carbon atomadjacent to disulphide bond that protects it from attack by thiolateanions, thus improving the in vivo stability of the linkage (40, 41).

Based on these data, IgG proteins can be modified with SMPT, which donot significantly affect the antigen binding property of the moleculesin the following conditions, even if they change the charge of theprotein in the reaction site.

In the present study the inventors investigated conjugating humanizedanti-FAP-IgGls with recNgA, using 2 different recNgA:mAb molar ratios of2.5 and 3.5, after derivatization using an SMPT:mAb molar ratio of 6,following conjugation protocols (36). Purification was performed by SizeExclusion chromatography on Sephacryl 5200 (37).

Under the described conditions, the immunotoxin is predominantly amixture of antibody linked to one or two toxin molecules, with thepresence of high molecular weight components (IgG linked to several RIPproteins), as well as free and polymeric RIPs (dimeric in the case ofrecNgA) and free antibody. Thus, a careful purification is thought to bedesirable to obtain a pure product.

Biochemical Characterization

Anti-FAP hu36-IgGl-recNgA immunotoxin conjugates were produced andcharacterized as follows:

-   -   Conjugate HPS131-001-1    -   Concentration 0.277 mg/ml    -   Drug:antibody ratio (DAR): 1.8    -   PM: 182 kDa    -   Purity: 87% (13% of free mAb)

In Vitro Activity Testing

Activity testing on conjugates prepared as described above was performedthough evaluation of RIP activity in rabbit reticulocyte cell-freelysate (RRL) assay (FIG. 9), and cytotoxic effect on cell cultures(FIGS. 10A and 10B).

The RRL assay results show that the anti-FAP hu36-IgG1-recNgA conjugates(HPS131-001-1) presented similar IC50 values as native Nigrin-b orrecNgA and were in the 3 μM range, showing that antibody conjugation didnot diminish the enzymatic activity of recNgA (see FIG. 9).

The cell cytotoxicity results show that, on HT1080 wild-type cells,conjugated antibody HPS131-001-1 displays only slight toxicity (if any)and only at highest concentration, naked anti-FAP hu36-IgG1 does nothave any effect, and recNgA shows cytotoxic effect only at 10⁻⁶M andafter 72h incubation (see FIG. 10A).

However, on FAP-expressing cells, HT1080-FAP, only HPS131-001-1conjugated anti-FAP antibodies strongly reduce HT-1080-FAP cellviability in the picomolar concentration range, with IC₅₀ values of 5 μM(see FIG. 10B).

These results show that: 1) anti-FAP:recNgA immunotoxins are highlyactive in vitro, being cytotoxic at picomolar range; 2) Activity ishighly specific to FAP-expression, since no significant effect wasobserved in HT1080-WT; 3) Anti-FAP hu36-IgG1 specificity for its targetis not affected by the conjugation to recNgA, neither is the enzymaticRIP activity of recNgA; 4) Activity is specific of the conjugatedanti-FAP hu36-IgG1, since no effect was observed with the naked IgG1; 5)Anti-FAP:recNgA immunotoxins are internalized, since non conjugatedrecNgA (lacking membrane binding domain) shows almost no cytotoxiceffect (IC₅₀>1 μM)(see FIG. 8).

In summary, anti-FAP:recNgA immunotoxins have the ability in vitro tospecifically recognize the target (FAP), to be internalized within thecytosol and release the recNgA effector moiety to actively inhibitribosomes, resulting in cytotoxicity IC₅₀ values within the picomolarrange.

In Vivo Evaluation of Anti-Tumoral Effect

Immunotoxin anti-FAP:recNgA has been tested in vivo in both cell-derivedand patient-derived xenograft mouse models for pancreas cancer. A doserange study was first performed to define the maximum tolerated dose innormal mice and each of these models: doses from 5 to 0.1 mg/kg wereadministrated intraperitoneally once a week during 3 weeks, and animalweight was monitored every 2 days to detect possible weight loss due totoxic effect of the immunotoxin. Results are presented in FIG. 13.

High doses (>0.5 mg/kg) induced hepatotoxicity in normal mice, while noFAP-dependent toxicity was observed after pathological analysis ofuterus and skeletal muscle, where low FAP expression has been described(Dolznig H., et al., Cancer Immun., 5:10,2005; Roberts E. W., et al., J.Exp.Med., 210:1137, 2013), nor in heart and kidney. Doses lower than 0.5mg/kg did not induce any detectable non-specific toxicity in cellline-derived orthotopic (FIG. 13) and patient-derived subcutaneous (FIG.14A) xenograft murine models of pancreas cancer.

In efficacy studies performed then at nontoxic doses from 0.5 to 0.1mg/kg, anti-FAP:recNgA immunotoxin, applied as single agent or incombination with Gemcitabine (240mg/kg), has shown no in vivoantitumoral efficacy in FAP (−) cell line derived orthotopic xenograftmurine models (not shown), while high in vivo antitumoral efficacy wasevidenced at a dose of 0.5 mg/kg in FAP (+) patient-derived subcutaneousxenograft murine models of pancreas cancer (FIG. 14B). When combinedwith Gemcitabine (150 mg/kg), it even showed 100% tumor growthinhibition and tumor regression.

Example 5 Cytolysins and Their Conjugation to Anti-FAP Antibodies

Tubulysins are recently discovered natural compounds isolated fromMyxobacteria, able to destabilize the tubulin skeleton, inducingapoptosis with a very high activity.

Leading to a fast, irreversible and strong change in the cellmorphology, tubulysins and their synthetic tetrapeptidic analogues, thecytolysins, are highly potent cell-killing agents (nM to pM activity).Tubulysin A inhibits tubulin polymerization in vitro with an IC₅₀ of0.75-1 μM, thus blocking the formation of mitotic spindles and inducingcell cycle arrest in G2/M phase. Tubulysins compete strongly withvinblastine through binding on the vinblastine binding site of tubulin.Furthermore they are stable in lysosome enriched cell fractions (45-48).

Amenable to conjugation, many different tubulysin/cytolysin derivativesare accessible by total synthesis in sufficient quantities forpreclinical and clinical development; functional groups in theirstructure can be adapted to several different linker technologies.

The cytolysins employed for conjugation studies were chosen from thegeneral structure shown above (formula IV). These structures exhibitactivity against different cancer cell lines (nM to pM range).

Various linker systems can be used and attached to either R² or R¹⁷position of the molecule.

The general outline of the cytolysin conjugates, including the vcPABAlinker and anti-FAP antibody, is shown in FIG. 11 (in the structuredepicted in FIG. 11, the attachment site of the cytolysin to the vcPABAlinker is at position R₁ or R₄—the R₁ and R₄ numbering system used inFIG. 11 differs from the R group numbering system used, e.g., in theclaims; it is intended that R₁ of FIG. 11 corresponds to R² in theclaims and that R₄ of FIG. 11 corresponds to R¹⁷ of the claims).

The vcPABA (valine-citrulline-PRBC) protease-cleavable linker has beenpreviously used in the ADC molecule Brentuximab Vedotine, developed bySeattle Genetics and Takeda, and recently approved by the FDA and EMEAas Adcetris® (2011, and Nov. 2012, respectively). In this ADC the vcPABAhas been coupled at its free NH2 to maleimide caproyl for thiol-basedconjugation on mAb (cAC10 anti-CD30 antibody). On the other side, vcPABAhas been conjugated through its COOH to the Auristatin cytotoxic drugfrom Seattle Genetics (MMAE). (see 49)

The present inventors have used this linker (maleimide caproyl-vcPABA)to conjugate anti-FAP antibodies through thiol-based reaction with themaleimide caproyl, and on the other end, to the cytolysin cytotoxicmolecules through its cyclic piperidine with vcPABA (R1 or R4 positionsof the cytolysin shown in FIG. 11).

Synthesis of Maleimido-val-cit-PABOCO-Tubulysin/Cytolysin-TAM461:

TAM461 (Tubulysin/Cytolysin): 30.0 mg (0.041 mmol)

-   -   DMF: 3 mL        TAM465 (Linker): 35 mg (0.045 mmol)    -   HOBt: 1.4 mg

DIPEA: 10 μL

TAM461 and TAM465 were dissolved in anhydrous DMF under dry conditionsand the resulting solution was treated with HOBt and DIPEA. The reactionwas stirred at RT for 18h. The reaction mixture was concentrated and theresulting oil was purified by column chromatography using 2-6% methanol:DCM to give 35 mg (64%) of TAM467 as a white solid. ESI-MS: m/z=1371[M+H].

Synthesis of Maleimido-val-cit-PABOCO-Tubulysin/Cytolysin-TAM470:

TAM470 (Tubulysin/Cytolysin): 0.07 mmol

-   -   DMF: 5 mL        TAM466 (Linker): 50 mg (0.065 mmol)    -   HOBt: 2.4 mg    -   DIPEA: 18 μL

TAM470 and TAM466 were dissolved in anhydrous DMF under dry conditionsand the resulting solution was treated with HOBt and DIPEA. The reactionwas stirred at RT for 18 h and then analysed with TLC, indicatingcompletion of reaction. The reaction mixture was concentrated and theresulting oil was purified with column chromatography using 4-12%methanol: DCM to give 56 mg of TAM471 (yield: 62%). ESI-MS: 1384.6[M+1].

In vitro activity testing is performed. Functional activity will beevaluated through microtubule inhibition assay, while cytotoxic activityis determined through crystal violet viability assay.

Generation of Cytolysin-Linker Derivatives

Different cytolysin-linker derivatives were synthesized according to thegeneral structure presented in FIG. 11, where vcPABA linker was addedeither in position R1 (TAM467, TAM551) or R4 (TAM471, TAM553, TAM558),alone or with ethylene-glycol spacer (EG; n=1 to 3), or substituted byethylene glycol groups (n=3) (TAM552). The respective chemicalstructures are presented in Table 4.

TABLE 4 Chemical structures of cytolysin -linker derivatives Mol.Product Code Wt.

TAM467 1370.7

TAM551 1356.7

TAM471 1384.7

TAM552 1198.5

TAM553 1499.8

TAM558 1603.9 Microtubule inhibition inhibition activity and cytotoxicactivity of each new derivative was evaluated through tubulinpolymerization inhibition assay (TPI; Tubulin Polymerization assay kit;Cytoskeleton, Cat. #BK011P), and cell proliferation arrest on HT1080cells (CPA; crystal violet). IC50 were calculated and results arepresented in Table 5.

TABLE 5 Microtubule inhibition activity and Cell Cytotoxicity activityof cytolysin-linker derivatives. IC₅₀ (TPI IC₅₀ (CPA Compound assay; μM)assay; nM) TAM467 (Linker in R1) 150 230-420 TAM551 (Linker in R1) ND 90TAM471 (Linker in R4; 14 17-42 vcPABA) TAM552 (Linker in R4; no 1.9 10vcPABA; 3EG) TAM553 (Linker in R4; 6 98 vcPABA; 1EG) TAM558 (Linker inR4; 1.9 98 vcPABA; 3EG) TAM334 (parental cytolysin; 2 0.3-0.6 no linker)Tubulysin A ND 0.04-0.2  Tubulysin A + linker ND  5-20 MMAE (SeattleGenetics) ND 0.1-0.6 DM1-DM4 (Immunogen) ND 0.01-0.1  (ND: Notdetermined)

In vitro activity of parental cytolysin TAM334 is within the same rangeof other payloads currently used for the generation of antibody-drugconjugates such as auristatins (MMAE) or maytansinoids (DM1-DM4). Asexpected and previously described for other compounds from the

Tubulysin A family, upon addition of linker, cell cytotoxic activity ofcytolysins was decreased with respect to the parental compound TAM334.In addition, TAM467 derivative was presenting significantly lowestactivity in both assays. All the derivatives were used in conjugation togenerate ADC molecules.

Conjugation and Chemical Characterization of ADCs

Each of the newly generated derivatives was conjugated to the anti-FAPhu36 following a non-site-specific conjugation method on cysteineresidues. To this aim, one batch of antibody was reduced and reactedwith each of the derivatives. Different TCEP ratios were tested to reachoptimal DAR of 3-4, less than 10% of free antibody and drug. Optimalconjugation conditions were as follows: TCEP=2.5 and 3.57 Thiol levelsEllmann's. Conjugates were then purified on G25 Sephadex and analysedthrough Size Exclusion Chromatography (SEC) to determine their purity,as well as Hydrophobic Interaction Chromatography (HIC) and Polymericliquid reversed-phase chromatography (PLRP) to determine DAR, content offree antibody and distribution profile of different ADC species (0-8drugs/mAb). Content of free drug was evaluated by UV detection method at280nm. Results of chemical analysis (SEC, HIC and

PRLP profiles) were determined for each ADC and for free antibody (datanot shown). Biochemical characteristics of the ADCs is shown in Table 6.

TABLE 6 Summary of chemical characteristics of the different ADCmolecules HIC SEC mAb free purity Free Lot Drug Conc. mAb DAR 280 nmDrug Volume HPS157- TAM471 1.195 10.1% 3.38 92% 0% ~5.8 mL 039-001 mg/mL(6.931 mg) HPS157- TAM551 1.332 22.4% 3.08 74% 0% ~5.8 mL 039-002 mg/mL(7.726 mg) HPS157- TAM552 1.319 5.1% 3.84 97% 0% ~5.8 mL 039-003 mg/mL(7.650 mg) HPS157- TAM553 1.305 7.0% 4.10 84% 0% ~5.8 mL 039-004 mg/mL(7.569 mg) HPS157- TAM558 1.332 5.8% 3.92 93% 0% ~5.8 mL 039-005 mg/mL(7.726 mg)

The various drugs produced different levels of aggregation. SpecificallyADC HPS157-039-002 (TAM551) showed highest level of aggregation alreadyat DAR=3.08, leaving 22.4% of unconjugated antibody. A preliminaryconjugation with TAM467 also showed high level of aggregation: at DAR3.27, SEC purity was already only 67% with 16% of free drug (data notshown). These data were suggesting that vcPABA linker in position R1 wasapparently less than optimal for this type of cytolysin molecule underthese conditions.

Target Binding of Conjugates

Anti-FAP hu36:TAM471 ADC binding to huFAP fusion protein was analysed byELISA, and binding to HT1080-FAP cells by FACS (FIG. 15). For

FACS analysis, compounds were incubated either at serial dilutions (FIG.15B) or at one dilution (FIG. 15C; 10 nM) and detected with ananti-human IgG-PE (V chain specific).

EC₅₀ values obtained in both assays showed no significant differencewith respect to naked anti-hu/moFAP hu36 antibody (FIG. 15A & 15B). Nobinding was observed in FAP(−) cells such as HT1080-wt and HEK293 cells(FIG. 15C).

FIG. 16 shows that ADC-471 (FIG. 16B) specifically binds and gets fullyinternalized after 90min in HT1080-FAP cells, similarly to nakedanti-FAP antibody (FIG. 16A). These results evidenced that conjugationdid not affect target specificity and affinity, or internalizationability of the anti-FAP hu36 IgG1.

Example 6 Evaluation of In Vitro Cytotoxic Activity and In VivoAnti-Tumoral Effect

Anti-FAP:cytolysin ADC candidates were evaluated in vitro throughproliferation arrest assay (crystal violet staining). Results arepresented in FIG. 17 and IC₅₀ values in Table 7. Anti-tumoral effect ofeach ADC candidate was evaluated in a patient-derived xenograft (PDX)mouse model for pancreas cancer (PAXF-736). This model was previouslyselected for FAP expression level and stroma expansion. ADC compoundswere administrated once a week intraperitoneally at 2.5 mg/kg. Tumorvolume and body weight were measured twice a week. Vehicle-treated andGemcitabine-treated (150 mg/kg) PDX mice were used as negative andpositive control groups, respectively. Results are shown in FIG. 18.

Location of vcPABA linker alone in R1 position (ADC-551) generatedconjugates with much less cytotoxic activity in vitro in comparison withconjugates utilizing the R4 position (ADC-471) (FIG. 17; Table 7) and noanti-tumoral activity in vivo (FIG. 18).

Increasing the number of ethylene-glycol groups as spacer to vcPABAlinker in R4 position (ADC-471 (n=0) versus ADC-553 (n=1) and ADC-558(n=3)) was shown to increase FAP-specific cytotoxic activity in vitro(FIG. 17) and anti-tumoral effect in vivo (FIG. 18). The TAM552conjugate (ADC-552), having a 3 ethylene glycol spacer, but no vcPABApresent in the linker was found to exhibit minimal or no in vivoanti-tumoral activity (data not shown). While ADC-471 and ADC-553 showedlow and no FAP-specific cytotoxic activity (10 nM and 100 nM IC₅₀ range,respectively) with no difference between HT1080-WT and FAP cells noranti-tumoral effect in vivo, ADC-558 presented a 1 nM range FAP-specificcytotoxic activity with a specificity ratio of 500 between FAP(+) andFAP(−) HT1080 cells, and a 40% tumor growth inhibition effect at 2.5mg/kg dose in PDX mouse model for pancreas cancer. No weight loss, nortoxic effect was observed for none of the candidates at this dose (notshown).

TABLE 7 IC₅₀ values obtained in Proliferation Arrest Assay (nM) CompoundHT1080-WT HT1080-FAP TAM334 1.04 0.77 ADC-471 (HPS-157-039-001) 5.610.33 ADC-551 (HPS-157-039-002) 964 552 ADC-553 (HPS-157-039-004) 90 108ADC-558 (HPS-157-039-005) 555 0.96

Further investigation was carried out using ADC-558. Maximum tolerateddose (MTD) was performed in normal mice and ADC-558 was found to benon-toxic within 2.5 to 25 mg/kg dose range with a weekly treatment for3 weeks. Doses from 20, 10, and 5 mg/kg were then administrated weeklyfor 4 weeks to a PDX mouse model (Panc185) with high FAP expressionlevel and stroma expansion to confirm tumor growth inhibition and fullregression efficacy of the ADC-558 conjugate.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety.

The specific embodiments described herein are offered by way of example,not by way of limitation. Any sub-titles herein are included forconvenience only, and are not to be construed as limiting the disclosurein any way.

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1. A conjugate having the formula I:A-(L-D)_(p)   (I) or a pharmaceutically acceptable salt or solvatethereof, wherein: A is an antibody that selectively binds FAP; L is alinker comprising a spacer; D is a drug comprising a cytolysin offormula IV:

wherein: R² is H or C₁-C₄ alkyl; R⁶ is C₁-C₅ alkyl; R⁷ is C₁-C₅ alkyl,CH₂OR¹⁹ or CH₂OCOR²⁰, wherein R¹⁹ is alkyl, R²⁰ is C₂-C₆-alkenyl,phenyl, or CH₂-phenyl; R⁹ is C₁-C₅ alkyl; R⁹ is H, OH, O-alkyl orO-acetyl; f is 1 or 2; R¹¹ has the following structure:

wherein R²¹ is H, OH, halogen, NH₂, alkyloxy, phenyl, alkyl amino ordialkyl amino; R¹⁶ is H or a C₁-C₆-alkyl group; R¹⁷ is CONHNHX and saidspacer is a —(OCH₂CH₂)_(n)— attached to R¹⁷ via a —C(O)X bridging group,wherein n=2, 3 or 4, and wherein X represents the bond between thespacer and R¹⁷; q is 0, 1, 2 or 3; and p is 1 to
 10. 2. The conjugate ofclaim 1, wherein A comprises heavy chain complementarity determiningregions 1-3 (CDRH1-3) and light chain complementarity determiningregions 1-3 (CDRL1-3) having the following amino acid sequences: (i)CDRH1: SEQ ID NO: 7; (ii) CDRH2: SEQ ID NO: 8; (iii) CDRH3: SEQ ID NO:9; (iv) CDRL1: SEQ ID NO: 10; (v) CDRL2: SEQ ID NO: 11; and (vi) CDRL3:SEQ ID NO:
 12. 3. The conjugate of claim 1, wherein L comprises anattachment group for attachment to A and a protease cleavable portion.4. The conjugate of claim 3, wherein L comprisesmaleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate.
 5. Theconjugate of claim 1, wherein R² is methyl, f=2, R⁶ is C₄ alkyl, R⁷ isC₃ alkyl, R⁹ is C₃ alkyl, R¹⁹ is O-alkyl, R²¹ is OH, q is 1, and R¹⁶ ismethyl.
 6. The conjugate of claim 1, wherein n=3.
 7. A method oftreating a tumor in a mammalian subject, wherein said tumor and/orstroma surrounding said tumor expresses fibroblast activation proteinalpha (FAP), said method comprising administering a therapeuticallyeffective amount of a conjugate of claim 1 to a subject in need thereof.8. The method of claim 7, wherein said conjugate is administeredsimultaneously, sequentially or separately with one or more otherantitumor drugs.
 9. The method of claim 8, wherein said one or moreother antitumor drugs comprise a cytotoxic chemotherapeutic agent or ananti-angiogenic agent or an immunotherapeutic agent.
 10. The method ofclaim 9, wherein said one or more other antitumor drugs compriseGemcitabine, Abraxane, bevacizumab, itraconazole, orcarboxyamidotriazole, an anti-PD-1 molecule or an anti-PD-L1 molecule.11. The method of claim 10, wherein said anti-PD-1 molecule oranti-PD-L1 molecule comprises nivolumab or pembrolizumab.
 12. The methodof claim 7, wherein the FAP expressing tumor is a solid tumor.
 13. Themethod of claim 12, wherein the solid tumor is an FAP expressing solidtumor of pancreatic cancer, breast cancer, melanoma, lung cancer, headand neck cancer, ovarian cancer, bladder cancer or colon cancer.
 14. Amethod of treating a fibroblast activation protein alpha (FAP)expressing inflammatory condition in a mammalian subject, comprisingadministering a therapeutically effective amount of a conjugate of claim1 to a subject in need thereof.
 15. The method of claim 14, wherein saidFAP expressing inflammatory condition is FAP expressing rheumatoidarthritis.
 16. A conjugate having the formula I:A-(L-D)_(p)   (I) or a pharmaceutically acceptable salt or solvatethereof, wherein: A is an antibody that selectively binds FAP; L is alinker; D is a drug having the structure:

wherein p is 1 to
 10. 17. The conjugate of claim 16, wherein L comprisesmaleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate.
 18. Theconjugate of claim 16, wherein A competes with the anti-FAP IgG1antibody having the heavy chain amino acid sequence of SEQ ID NO: 3 andthe light chain amino acid sequence of SEQ ID NO: 4 for binding toimmobilized recombinant human FAP.